title: Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament.
abstract: OBJECTIVE
  Molecular mechanism of ossification of the posterior longitudinal ligament (OPLL) remains unclear. This study was to investigate different expressions of PERK between the spinal ligament fibroblasts from OPLL patients and non-OPLL patients, and demonstrate knockdown of PERK protein expression by RNA interference inhibiting expression of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) in the cells from OPLL patients.
  
  
  METHODS
  Spinal ligament cells were cultured using tissue fragment cell culture and identified by immunocytochemistry and immunofluorescence. The mRNA expression of osteoblast-specific genes of OCN, ALP and COL I was detected in the cells from OPLL and non-OPLL patients by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of PERK was detected by Western blotting. And then, after 72 h, when RNA interference against PERK was performed on the cells from OPLL patients, expression of the osteoblast-specific genes was compared again between the transfection group and non-transfection group.
  
  
  RESULTS
  Spinal ligament fibroblasts were observed 7-10 days after cell culture. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The mRNA expressions of OCN, ALP and COL I and protein expression of PERK in the cells from OPLL patients were significantly greater than those from non-OPLL patients. In addition, knockdown of PERK protein expression inhibited the mRNA expressions of OCN, ALP and COL I remarkably in the transfection group compared with the non-transfection group, at 72 h after RNA interference targeting PERK was performed on the cells from OPLL patients.
  
  
  CONCLUSIONS
  The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and PERK-mediated ER stress might be involved in development of OPLL.
authors.affiliation: Department of Spine Surgery, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai, 200003, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.forename: Xinwei
authors.initials: X
authors.lastname: Wang
authors.forename: Haisong
authors.initials: H
authors.lastname: Yang
authors.forename: Jinhao
authors.initials: J
authors.lastname: Miao
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Liu
authors.forename: Deyu
authors.initials: D
authors.lastname: Chen
mesh_terms: D000328:Adult; D015153:Blotting, Western; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D017846:Longitudinal Ligaments / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D017887:Ossification of Posterior Longitudinal Ligament / Q000235:genetics / Q000378:metabolism*; D010012:Osteogenesis; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D015854:Up-Regulation; D019892:eIF-2 Kinase / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D012333:RNA, Messenger; C460640:EIF2AK3 protein, human; D019892:eIF-2 Kinase